Co-delivery of gentiopicroside and thymoquinone using electrospun m-PEG/PVP nanofibers: In-vitro and In vivo studies for antibacterial wound dressing in diabetic rats.

Nanofibers (NFs) provide several delivery advantages like their great flexibility and similarity with extracellular matrix (ECM) which qualify them to be the unique model of a wound dressing. NFs could create mats of polymeric matrix loaded with an active agent enhancing its solubility and stability. In our study, Gentiopicroside (GPS) and Thymoquinone (TQ) loaded in NFs polymeric mats composed of coblended polyvinyl pyrrolidine (PVP) and methyl ether Polyethylene glycol (m-PEG) were fabricated via electrospinning technique. A morphological study using Scanning Electron Microscopy (SEM) was performed for all formulae as well as in vitro release study using High-performance Liquid chromatography (HPLC) for sample analysis. The optimized formula (F3) was chosen for further assays using Fourier-Transform Infrared Spectroscopy (FTIR), and Differential Scanning Calorimetry (DSC). Study of the antibacterial effect, and in vivo healing action for diabetic infected wounds to quantify Tumor necrosis factor-alpha and Cyclooxygenase-2 were also investigated. F3 achieved the highest % cumulative release (99.79 ± 6.47 for GPS and 96.89 ± 6.87 for TQ) at 60 min, and a smaller diameter (200 nm) showing significant anti-bacterial effects with well-organized skin architecture demonstrating great healing signs. Our results revealed that m-PEG/PVP NFs mats loaded with GPS and TQ could be considered an optimal wound care dressing.

Carbon quantum dots as a sensitive fluorescent probe for quantitation of pregabalin; application to real samples and content uniformity test.

A novel optical nano-sensor for the detection of pregabalin (PG) in its pharmaceutical (Lyrica®) capsules and biological samples was reported. For the fabrication of highly fluorescent carbon quantum dots (CQDts), a simple green hydrothermal approach was described, and ascorbic acid (AA) was used as a carbon source. The obtained CQDts were confirmed by spectroscopic characterization such as transmission electron microscopy (TEM) and Fourier-transform infrared (FTIR) spectra. The synthesized CQDts were capped by alcohol to form yellow emitters, showing strong fluorescent emission at 524 nm, and excitation at 356 nm. The method is based on fluorescence quenching of CQDts in the presence of PG. The proposed analytical method was validated according to ICH guidelines. PG was successively assayed in the concentration range of 4.0 to 100 µg/ml). The detection and quantitation limits were 1.12 and 3.39 µg/ml, respectively. The proposed method could be used in both quality control and pharmacokinetic research for the studied drug.

In Vitro and In Vivo Antitumor Activity of Indolo[2,3-b] Quinolines, Natural Product Analogs from Neocryptolepine Alkaloid.

Neocryptolepine (5-methyl-5H-indolo[2,3-b] quinoline) analogs were synthesized and evaluated in vitro and in vivo for their effect versus Ehrlich ascites carcinoma (EAC). The analogs showed stronger cytotoxic activity against EAC cells than the reference drug. The in vivo evaluation of the target compounds against EAC-induced solid tumor in the female albino Swiss mice revealed a remarkable decrease in the tumor volume (TV) and hepatic lipid peroxidation. A noticeable increase of both superoxide dismutase (SOD) and catalase (CAT) levels was reported (p < 0.001), which set-forth proof of their antioxidant effect. In addition, the in vitro antioxidant activity of the neocryptolepine analogs was screened out using the DPPH method and showed promising activities activity. The histopathological investigations affirmed that the tested analogs have a remarkable curative effect on solid tumors with minimal side-effect on the liver. The study also includes illustrated mechanism of the antitumor activity at the cell level by flow cytometry. The cell cycle analysis showed that the neocryptolepine analogs extensively increase the aggregation of tumor cells in three phases of the cell cycle (G0/G1, S and G2/M) with the emergence of a hypo-diploid DNA content peak (sub-G1) in the cell cycle experiments, which is a clear-cut for the apoptotic cell population. Furthermore, the immunological study manifested a significant elevation in splenic lymphocyte count (p < 0.001) with the elevation of the responsiveness of lymphocytes to phytohemagglutinin (PHA). These results indicate that these naturally-based neocryptolepine alkaloids exhibit marked antitumor activity in vivo and represent an important lead in the development of natural-based anticancer drugs.

Application of MEKC and UPLC with Fluorescence Detection for Simultaneous Determination of Amlodipine Besylate and Bisoprolol Fumarate.

OBJECTIVE: Two chromatographic methods were described for simultaneous determination of the antihypertensive drugs amlodipine besylate (AML) and bisoprolol fumarate (BIS). METHODS: Method I applies micellar electrokinetic capillary chromatography using a deactivated fused silica capillary (25 cm effective length × 50 µm internal diameter). The background electrolyte consisted of 0.01 M borate buffer (pH 9.2) containing 0.025 M sodium dodecyl sulphate and methanol in the ratio of 80:20 (v/v). Valsartan (VAL) was used as an internal standard. Diode array detector was set at 238, 224, and 210 nm for measuring AML, BIS, and VAL, respectively. Method II involves using ultra-performance liquid chromatography with fluorescence detection. Zorbax SB-C8 column (2.1 × 100 mm, 1.8 µm particle size) was used with isocratic elution of the mobile phase composed of 0.1% trifluoroacetic acid, acetonitrile, and methanol in the ratio of 55:35:10 (v/v) at a flow rate of 0.6 mL/min. Fluorescence detection was done using excitation wavelengths 230 and 370 nm and emission wavelengths 305 and 450 nm for BIS and AML, respectively. Validation parameters were carefully studied including linearity, ranges, precision, accuracy, robustness, detection, and quantification limits. RESULTS: Method I showed good linearity over the range 10-100 µg/mL for both dugs. Method II's linear ranges were 0.001-0.1 and 0.02-1 µg/mL for BIS and AML, respectively. CONCLUSION: The proposed methods were successfully validated and applied for assay of the studied drugs in their fixed-dose combination tablets. HIGHLIGHTS: To the best of our knowledge, this study suggests the first electro-chromatographic and LC with fluorescence detection methods for simultaneous determination of amlodipine and bisoprolol.

Fluorescence spectroscopy for determination of dapagliflozin in pure form and its tablets formulation; Application to content uniformity testing.

A new, valid method was developed for quantitative spectrofluorimetric analysis of dapagliflozin (DGF) in its pure form and its commercially available tablets (Farxiga®). The proposed analytical method based on measurement of fluorescence intensity for DGF at 303 nm after excitation at 278 nm. Various experimental parameters influencing the fluorescence of DGF were examined to produce the optimal conditions. DGF was successively assayed in concentration range of (100-1000 ng mL-1) with lower detection limit (LOD) of 26.49 and quantitation limit (LOQ) was 79.48 ng mL-1. The suggested method was validated according to ICH guidelines for the estimation of DGF in its pure form and its new dosage form with percent recovery of 100.43 ±â€¯1.15. The proposed method was adapted to examine DGF in content uniformity testing according to United States Pharmacopeia guidelines. This method can be used in routine analysis of DGF in quality control lab.

New spectrofluorimetric analysis of empagliflozin in its tablets and human plasma using two level full factorial design.

An efficient, accurate and sensitive spectrofluorimetric method was developed for analysis of empagliflozin (EGF) in pure form, dosage form and human plasma. The proposed procedure was based on formation of yellow fluorescent product between benzofurazan reagent and empagliflozin in slightly alkaline medium that is measured at 521 nm, when excitation at 455 nm. The present study was validated according to ICH guidelines and bioanalytical validated according to US-FDA guidance. The fluorescence intensity-concentration plot was linear over the range of 50-1000 ng ml-1 with limit of detection (LOD) and quantitation (LOQ) of 15.55 and 46.63 ng ml-1, respectively. The correlation (r) and determination (r2) coefficient was 0.9998 and 0.9997, respectively. Due to high sensitivity and selectivity of the proposed method, it is successfully used for analysis of empagliflozin in its dosage form and human plasma with good recoveries of 98.89% and 98.70%, respectively, without any interfering from matrix components. The corresponding regression equation, Y = 0.756X + 141.93, (r2 = 0.9994) for spiked plasma sample. Two level full factorial designs were used to study different experimental parameters that affect the reaction product and to get the optimum method conditions. The suggested method can be used in quality control lab as well as in pharmacokinetic studies of empagliflozin.

Second-derivative synchronous spectrofluorimetric assay of dapagliflozin: Application to stability study and pharmaceutical preparation.

A highly accurate, simple and sensitive spectrofluorimetric analytical method for dapagliflozin (DGF) quantitation was developed. The proposed method was successively applied to DGF analysis in both its pure and pharmaceutical dosage forms. This method was developed to investigate DGF stability in its degradation products, as laid out in International Council for Harmonisation (ICH) rules. Kinetics of alkaline degradation of DGF was also calculated. The half-life time (t1/2 ) of the reaction was 75.32 min. An alkaline degradation pathway was described. The present study involved measurement of the second-derivative synchronous fluorescence intensity of DGF at Δλ = 30 nm. Peak amplitude was measured at 322 nm. Linear range of the calibration curve was 0.1-1.0 µg ml-1 . Lower detection and quantitation limits were 0.023 and 0.071 µg ml-1 , respectively, and indicated good sensitivity of the proposed method. Mean per cent recovery was 99.78 ± 1.78%. The proposed analytical approach was successfully applied to DGF in the quality control laboratory and would be suitable as a stability-indicating assay.

New spectrofluorimetric analysis of dapagliflozin after derivatization with NBD-Cl in human plasma using factorial design experiments.

A new validated spectrofluorimetric method was proposed for dapagliflozin (DGF) analysis in bulk, plexin its commercially available tablets and in spiked human plasma. The proposed spectrofluorimetric method depended on the formation of a fluorescent complex soluble in organic liquids by a substitution reaction between 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reagent and DGF in aqueous buffered solution at pH 7. The fluorescence intensity was measured at 522 nm after excitation at 453 nm. The high selectivity of the proposed method allowed analysis of DGF in dosage form and human plasma samples with average recovery values of 99.84 ± 1.38% and 98.71 ± 1.80%, respectively, without any interference from matrix components. The calibration range was 50-1000 ng ml-1 . The limit of detection (LOD) and limit of quantitation (LOQ) were 14.24 ng ml-1 and 43.14 ng ml-1 , respectively. The estimated relative standard deviation values were lower than 2.0%, this showed the excellent precision at both levels. Factorial design was used to get the optimum method conditions for the analysis of the resulting DGF fluorescence complex in different matrices. The proposed method could be used in routine analysis of DGF in quality control laboratories. Also, it could be used to assay DGF in human plasma and be applied for pharmacokinetic investigation of DGF.

Determination of Ibuprofen and Phenylephrine in Tablets by High-Performance Thin Layer Chromatography and in Plasma by High-Performance Liquid Chromatography with Diode Array Detection.

Two chromatographic methods (high performance thin layer chromatography (HPTLC) and high performance liquid chromatography-diode array detector (HPLC-DAD)), were addressed for the analysis of a mixture consisted of phenylephrine hydrochloride and ibuprofen in two forms bulk and their combined dosage form. This binary mixture is considered to be a challenging one as the two drugs differ greatly in their chemical and physical properties. Not only this affects their simultaneous analysis, but also hinders their simultaneous extraction from biological fluids as plasma. That is the reason the literature lacks any report for the simultaneous extraction and analysis of these drugs from biological fluids. The concentration ranges of both drugs were 0.1-2.5 µg/spot and 0.1-100 µg/mL by HPTLC and HPLC, respectively. Not only was the HPLC-DAD method applied to the investigated drugs determination in pharmaceutical preparations, but also in spiked human plasma. Extensive study was conducted to optimize their simultaneous extraction from plasma as it was a crucial step for the in vivo analysis. The results obtained by proposed methods and a reference one were statistically comparable by analysis of variance test. No significant difference was recorded between the mean percent levels determined by the proposed methods and the reference one.

Application of Capillary Zone Electrophoresis Coupled with a Diode Array Detector (CZE-DAD) for Simultaneous Analysis of Ibuprofen and Phenylephrine.

Background: A validated method based on capillary zone electrophoresis coupled with a diode array detector (CZE-DAD) was investigated for analyzing binary mixture of ibuprofen (IBU) and phenylephrine (PHE) in their bulk and combined dosage form. Objective: This binary mixture is a challenging one as IBU is acidic and PHE is alkaline, which may affect their simultaneous analysis using CZE. The literature lacks any CZE report for IBU and PHE simultaneous analysis. Methods: Fused silica capillary (85 cm × 75 µm id) was used, and the electrolyte was a 50 mM borate buffer adjusted to pH 11 with 0.5 M NaOH. Results: The concentration ranges were 5-200 and 5-100 µg/mL for IBU and PHE, respectively, using CZE. High efficiency was achieved (N > 92990). Reasonable migration time (tm) was attained (tm< 8.5 min). Conclusions: Although the results obtained by the proposed CZE method and reported HPLC method were statistically comparable, the proposed method showed lower linearity ranges, higher efficiency, and a more reasonable run time. Highlights: CZE-DAD was used for the analysis of IBU and PHE in bulk and tablets, as no report was found for their determination using CZE. Binary mixture is challenging due to differences in chemical and physical properties. A detailed discussion of electrophoretic parameters optimization is included. Confirmation of peak purity was attained using DAD.

Simple protein precipitation extraction technique followed by validated chromatographic method for linezolid analysis in real human plasma samples to study its pharmacokinetics.

Fast and sensitive HPLC method was developed, optimized and validated for quantification of linezolid (LNZ) in human plasma using guaifenesin as an internal standard (IS). Analyte and IS were extracted from plasma by simple protein precipitation extraction technique using methanol as the precipitating solvent. The pretreated samples were injected in a mobile phase formed of acetonitrile:water:methanol (20:70:10v/v/v) in an isocratic mode at a flow rate of 1.5mL/min with UV detection at 251nm. Separation was done using Aglient ODS C18. The method showed linearity in the range of 0.75-50µg/mL with correlation coefficients equals to 0.9991. Precision and accuracy were in conformity with the criteria normally accepted in bio-analytical method validation. The RSDs for intra- and inter-day assays were <3.56 and 4.63%, respectively. The intra- and inter-day accuracies were 94.67-98.28% and 91.25-96.18%, respectively. The mean absolute recoveries ranged from 92.56±1.78 to 95.24±2.84. According to stability results, LNZ was stable in human plasma during the storage and analysis. LNZ a pharmacokinetic behavior was studied by applying the proposed analytical method.

High-performance liquid chromatography with diode array detection method for the simultaneous determination of seven selected phosphodiesterase-5 inhibitors and serotonin reuptake inhibitors used as male sexual enhancers.

This work presents a simple, sensitive and generic high-performance liquid chromatography with diode array detection method for the simultaneous determination of seven drugs prescribed for the treatment of erectile dysfunction and premature ejaculation. Investigated drugs include the phosphodiesterase-5 inhibitors: sildenafil, tadalafil, and vardenafil, in addition to the selective serotonin reuptake inhibitors: dapoxetine, duloxetine, fluoxetine, and paroxetine. The drugs were separated using a Waters C8 column (4.6 × 250 mm, 5 µm) with the mobile phase consisting of phosphate buffer pH 3, acetonitrile and methanol in the ratio 60:33:7. The flow rate was 1.2 mL/min, and quantification was based on measuring peak areas at 225 nm. Peaks were perfectly resolved with retention times 3.3, 3.9, 6.4, 7.5, 9.5, 10.7, and 13.4 min for vardenafil, sildenafil, paroxetine, duloxetine, dapoxetine, fluoxetine, and tadalafil, respectively. The developed method was validated with respect to system suitability, linearity, ranges, accuracy, precision, robustness, and limits of detection and quantification. The proposed method showed good linearity in the ranges 5-500, 2-200, 2-200, 3-300, 1.5-150, 2-200, and 2-200 µg/mL for sildenafil, tadalafil, vardenafil, dapoxetine, duloxetine fluoxetine, and paroxetine, respectively. The limits of detection were 0.18-0.38 µg/mL for the analyzed compounds. The applicability of the proposed method to real life situations was assessed through the analysis of commercial tablets, and satisfactory results were obtained.

New valid spectrofluorimetric method for determination of selected cephalosporins in different pharmaceutical formulations using safranin as fluorophore.

A new validated spectrofluorimetric method has been developed for the determination of some cephalosporins namely; cefepime, cefaclor, cefadroxil, cefpodoxime and cefexime. The method was based on the reaction of these drugs with safranin in slightly alkaline medium (pH 8.0), to form ion-association complexes. The fluorescent products were extracted into chloroform and their fluorescence intensities were measured at 544-565 nm after excitation at 518-524 nm. The reaction conditions influencing the product formation and stability were investigated and optimized. The relative fluorescence intensity was proportional to the drug concentration in the linear ranges of 0.15-1.35, 0.35-1.25, 0.35-1.25, 0.20-1.44 and 0.20-1.25 µg/mL for cefepime, cefaclor, cefadroxil, cefpodoxime proxetil and cefexime, respectively. The detection limits were 40, 100, 100, 60 and 70 ng/mL, respectively. The performance of the developed method was evaluated in terms of Student's t-test and variance ratio F-test to find out the significance of proposed methods over the reference spectrophotometric method. Various pharmaceutical formulations were successfully analyzed using the proposed method and the results were in good agreement with those of the previously reported methods.

The use of laser-induced fluorescence or ultraviolet detectors for sensitive and selective analysis of tobramycin or erythropoietin in complex samples.

Complex samples analysis is a challenge in pharmaceutical and biopharmaceutical analysis. In this work, tobramycin (TOB) analysis in human urine samples and recombinant human erythropoietin (rhEPO) analysis in the presence of similar protein were selected as representative examples of such samples analysis. Assays of TOB in urine samples are difficult because of poor detectability. Therefore laser induced fluorescence detector (LIF) was combined with a separation technique, micellar electrokinetic chromatography (MEKC), to determine TOB through derivatization with fluorescein isothiocyanate (FITC). Borate was used as background electrolyte (BGE) with negative-charged mixed micelles as additive. The method was successively applied to urine samples. The LOD and LOQ for Tobramycin in urine were 90 and 200ng/ml respectively and recovery was >98% (n=5). All urine samples were analyzed by direct injection without sample pre-treatment. Another use of hyphenated analytical technique, capillary zone electrophoresis (CZE) connected to ultraviolet (UV) detector was also used for sensitive analysis of rhEPO at low levels (2000IU) in the presence of large amount of human serum albumin (HSA). Analysis of rhEPO was achieved by the use of the electrokinetic injection (EI) with discontinuous buffers. Phosphate buffer was used as BGE with metal ions as additive. The proposed method can be used for the estimation of large number of quality control rhEPO samples in a short period.

Validated spectrofluorimetric method for determination of selected aminoglycosides.

New, sensitive, and selective spectrofluorimetric method was developed for determination of three aminoglycoside drugs in different dosage forms, namely; neomycin sulfate (NEO), tobramycin (TOB) and kanamycin sulfate (KAN). The method is based on Hantzsch condensation reaction between the primary amino group of aminoglycosides with acetylacetone and formaldehyde in pH 2.7 yielding highly yellow fluorescent derivatives measured emission (471 nm) and excitation (410 nm) wavelengths. The fluorescence intensity was directly proportional to the concentration over the range 10-60, 40-100 and 5-50 ng/mL for NEO, TOB and KAN respectively. The proposed method was applied successfully for determination of these drugs in their pharmaceutical dosage forms.